Construction Of Recombinant HBV Replication Cell Line With Stable Expression Of Luciferase And HBV Susceptible Cell Line

Chronic viral hepatitis B caused by HBV and its related diseases seriously endanger human health.Persistent infection of HBV is an important risk factor for developing cirrhosis and liver cancer.Strengthening the study of HBV molecular biology will provide an important basis for guiding the clinical treatment and discovery of new drugs,and will also help reduce the morbidity and mortality of HBV.In this study,we constructed recombinant HBV replicating cell lines and HBV susceptible cell lines,which provided a favorable tool for the study of HBV infection model and the receptor inhibitors.Part 1 Construction of recombinant HBV replication cell line stably expressing luciferaseObjective: Construct a recombinant HBV vector carrying the recombinant luciferase expression tag.When the hepler plasmid exists,it still has the ability to replicate and can form a complete HBV particles.Methods:1 The recombinant HBV plasmid with SecNluc tag was constructed by pTRE-HBV plasmid expressing wild-type HBV,with the secNluc tag was expressed in the XhoI-BsrGI segment of HBV S region HBs Ag.2 Construction of HBV helper plasmid pcDNA3.1(-)Bsd-CH3142 with resistance to blasticidin.3 The recombinant plasmid pTRE-HBV-S-NLuc expressing hygromycin resistant gene and HBV helper plasmid pcDNA3.1(-)Bsd-CH3142 with the blasticidin resistance gene were cotransfected into HepG2-TA2-7 cell lines with a ratio of 1:1.HBV replication cell clones stably expressing luciferase were selected by using the common pressure of neomycin,hygromycin and blasticidin.4 HBV DNA from cell supernant of the recombinant HBV cell clones was detected by fluorogenic quantitative PCR,homotheticly,the luciferase content was detected by Nano-Glo Luciferase Assay System kit.The cell clones with higher expression level of HBV DNA and luciferase were screened out for further detection.5 Using Native Western Blot to screen the formation of HBV core protein particles.6 Using southern blot to screen HBV DNA formation in cell clones.7 Transmission electron microscopy(TEM)was used to observe the formation of virus particles.Results:1 The recombinant plasmid pTRE-HBV-S-NLuc expressing hygromycin resistant gene and HBV helper plasmid pcDNA3.1(-)Bsd-CH3142 with the blasticidin resistance gene were cotransfected into HepG2-TA2-7 cell lines with a ratio of 1:1.HBV replication cell clones stably expressing luciferase were selected by using the common pressure of neomycin,hygromycin and blasticidin.36 clones were selected to continue to culture,numbered HBV-SNLuc1-36.Using fluorogenic quantitative PCR and Nano-Glo Luciferase Assay System kit detected the expression level of HBV DNA and the luciferase.After analysis and comparison,the 6 cell clones with high expression level of HBV DNA and luciferase were screened out,which were HBV-SNLuc(1,12,13,24,35,36).2 Native Western Blot showed the 6 cell clones and the control cell HepG2.117 all had the ability of the core protein particle formation.The results demonstrated that HBV-SNluc cells can produce core protein particles complete.3 Southern blot showed the 6 cell clones and the control cell Hep G2.117 all had the ability for the formation of loose ring,double stranded and single stranded HBV DNA.The results showed that HBV-SNluc cells were able to produce HBV replication intermediates,and had strong replication ability.4 After the above tests,we determined that HBV-SNLuc-35 was the bestcell line,which not only secreted higher HBV particles,but also expressed high levels of luciferase.5 The results of transmission electron microscopy(TEM)showed that theHBV-SNLuc-35 cell clones can produce virus particles similar to those of control group HepG2.117 cells.Conclusion: Using HepG2 cells construct HBV replication cell line stably expressing luciferase.A cell line HBV-SNLuc-35 with high expression of luciferase and recombinant HBV was screened after a series of studies.Part 2 Study on HBV susceptible cell line by QSG-7701 stably expressing NTCPObjective: In view of the fact that QSG-7701 cells can better support the replication of HBV,construct the QSG-7701 NTCP cell lines,which can express the stable level of sodium taurocholate cotransport polypeptide(NTCP)by using QSG-7701 cell.Methods:1 The expression of NTCP gene with Flag tag plasmid pAlb containing albumin promoter,in which inserts a hygromycin resistance gene cassette,the final plasmid pAlbHyg-NTCP-Flag with hygromycin resistance driven by albumin promoter to express NTCP.2 The plasmid pAlbHyg-NTCP-Flag expressing NTCP was transfected into QSG-7701 cell lines.The QSG-7701 NTCP cell lines,whicn can stably expressing NTCP on the cell membrane surface was observed by immunofluorescence assay.The cell clones with high expression of NTCP were selected.3 The laboratory constructed HBV replication cell line TC-3-1 was collected,which can stable express TC-FIAsH fluorescence labele.The recombinant HBV virus concentrated in cell supernatant infect QSG-7701 NTCP cells and he control groups: 293 T cells,QSG-7701 cells.The distribution of FIAsH fluorescence was observed by biarsenical dye and DAPI,which was proved that HBV can bind to NTCP on the liver cell membrane.4 The recombinant HBV stable expressing luciferase cell line infects thecell line that stably expresses NTCP.The susceptibility of NTCP to HBV was obsevered by multiple luciferase assay.Results:1 pAlbHyg-NTCP-Flag plasmid was transfected into QSG-7701 cells,and 12 cell clones were selected by using the pressure of hygromycin.The expression of NTCP on the cell membrane was detected by immu nofluorescence,4 cell clones with high expression were screened,which were named QSG-7701NTCP1~4 respectively.The expression of QSG-7701NTCP2 in the control group was the highest,while in the control gr oup,only QSG-7701;the expression of NTCP on the cell membrane su rface with the QSG-7701NTCP2 was highest,while the control group Q SG-7701 can only obsevered the weak fluorescence.2 QSG-7701NTCP2,293 T and QSG-7701 were infected by the recombinant HBV of fluorescent labeled TC-FIAsH,Using the biarsenical dye technique and DAPI detection can be observed that the experimental group had strong fluorescence,the control group(QSG-7701 cells)can be observed the faint fluorescence,while the other control group can not be observed the fluorescence.3 Virus infection by HBV-SNLuc-35 cell supernatant infect the QSG-7701NTCP1~4 and QSG-7701 cell lines.The expression level of luciferase was observed to increase from fourth days and last 4-5 days by multiple luciferase assay,and they were all higher than the expression in QSG-7701 cell lines and the QSG-7701NTCP4 was the highest.This confirmed that the susceptibility of QSG-7701 NTCP cell line to HBV.Conclusions: Construct HBV receptor cell line stably expressing NTCP by using QSG-7701 cell,a cell line QSG-7701NTCP4 was screened with high susceptibility after the experimental study of infection.